When diagnosing cancer, cellular features in biopsy samples are taken into account such as, the degree of variability of cancer cell size and shape, the proportion of actively dividing cells and invasion into neighbouring structures. Commonly used histological stains are haematoxylin (primary stain) and eosin (counterstain) which differentially label subcellular elements. Other diagnostic methods employ antibodies to particular diagnostic molecules within (via intracellular epitopes) or on the surface of cells or tissues (via extracellular epitopes) which can be made visible for microscopic analysis eg, carcino-embryonic antigen (CEA). Some specific examples are discussed below.
Prostate Cancer
The incidence of prostate cancer in the Western world is increasing at an alarming rate, having more than doubled in the past five years. It has the highest incidence of any neoplasm, is second only to lung cancer as the most common cause of cancer death in men worldwide, and is the leading cause of death in Australia [1]. Benign prostatic hyperplasia (BPH) is common in men over 50 and is a possible precursor of prostatic intraepithelial neoplasia (PIN), itself a precursor to prostate cancer. Postmortem studies indicate that 70% of men have malignant cells in their prostate by the time they reach 80 [2]. This disease is characterised by a striking racial variation and is most prevalent in African-Americans, intermediate in Caucasians, slightly lower in Latinos, and least prevalent in Asians. In the latter group, it is nevertheless the most rapidly increasing form of neoplasm. Until recently, it was not clear if these differences were due to racial genetic variation or diet. Studies have now shown that diet is a primary influencing factor [3].
Current Diagnosis and Treatment of Prostate Cancer
Despite the gravity of this condition, diagnostic methods are few and imprecise. Current methods for assessing prognosis such as digital rectal examination (DRE), ultrasound, prostatic acid phosphatase levels, androgen ablation, prostate specific antigen (PSA) density, PSA velocity, PSA age-specific reference ranges and Gleason histopathological grading, can fail to provide reliable predictive information regarding the clinical outcome of prostate cancer [4]. For instance, studies have shown that DRE results in a 36.9% false negative rate [5]. PSA is a 33-kDa serine protease that is associated with a number of tissues besides prostate [6], is up-regulated by androgens, glucocorticoids and progestins and is thought to be involved in the regulation of growth factors. Unfortunately, serum PSA levels have an incidence of 23% false negative and 36.7% false positive diagnoses [6]. It has even been suggested that more than half of new screen-detected cases are in fact false positives [7]. Attempts to improve screening methods by the introduction of additional tests such as PSA density, velocity, and age-specific reference ranges has been equivocal. One study has shown that applying an age-specific PSA reference range that increases the upper limit of normal PSA to 4.5 ng/mL results in the failure to detect a substantial number of clinically significant cancers [8]. Given this uncertainty, prostate biopsy is often performed to confirm malignancy but this test also has a highly unsatisfactory 23% incidence of false-negative diagnosis [9].
Treatment selection is largely dependent on clinical staging based on microscopic analysis of tissue sections [10]. This technique depends on judgment and considerable experience in relating histological appearance to clinical outcome. Unfortunately, prostate cancer tissue is notoriously heterogeneous and a vital diagnostic feature may easily be missed in the section being examined. To further complicate the situation, there have been no randomised and controlled trials to examine the outcomes of surgery and radiotherapy [2]. Treatment choices include radical prostatectomy, radiation therapy, androgen deprivation and “watchful waiting”. A definitive answer to the question of “watchful waiting” versus radical intervention awaits the conclusion of the prostate cancer intervention-versus-observation trial [11]. The consequences to the patient of these decisions are serious. Radical prostatectomy for instance, often results in incontinence, impotence, bladder neck stricture and depression [12]. Clearly, improved markers that reliably differentiate between benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), atypical adenomatous hyperplasia (AAH) and prostatic cancer are urgently needed.
The Role of P2X Receptors in Cancer
Neurotransmitters such as noradrenalin and acetylcholine act not only in the synapse and neuromuscular junction but also on transmitter-specific cell receptors in a wide variety of tissues and organs. These receptors are pore-like transmembrane channels that introduce ions into the cell. Adenosine triphosphate (ATP), best known as the molecular currency of intracellular energy stores, was first proposed as a peripheral neurotransmitter based on its ability to contract smooth muscle [13]. ATP acts in the same manner as other neurotransmitters and can activate both the (relatively slow) G protein-coupled tissue receptors (P2Y), the more recently characterised (fast) ligand-gated purinergic (P2X1-7) ion channels and can also act as a co-transmitter. Despite its relatively recent discovery, it is likely that the purinergic transmitter system developed very early in evolution [14].
There are currently 7 genetically distinct P2X receptor subtypes. They are as widely distributed as receptors of the cholinergic and adrenergic systems and are found in most mammalian cells [14]. These receptors constitute a new class of fast-response, membrane-bound, ligand-gated, calcium-permeable, cation-selective channels that are activated by extracellular ATP from nerve terminals or a local tissue source [15–18]. They are predominantly permeable to calcium ions but also admit other cations, such as potassium and sodium, thereby mediating depolarisation [19]. For instance, in lung epithelia, P2X channels stimulate Cl− channel up-regulation, K+ secretion and inhibit Na+ absorption (21). ATP can stimulate both DNA synthesis and cell proliferation via the up-regulation of the P2X receptors [14]. This function is linked to stimulation of phospholipase C and ionic calcium release from inositol-phosphate-sensitive intracellular stores, as well as other signal transduction pathways. These actions are potentiated by the synergistic action of ATP with polypeptide growth factors [20]. The influx of calcium through the P2X receptors also triggers the secretion of other neurotransmitters, serves as a signal for the activation of calcium-dependent potassium channels, inactivates other calcium channel types, regulates endocytotic retrieval of synaptic vesicle membranes, enhances the synthesis of neurotransmitters, regulates pools of synaptic vesicles available for secretion and triggers several forms of synaptic plasticity. The variety of responses to a single stimulation of P2X receptors suggests there are many calcium-activated pathways [21].
Extracellular ATP, acting via the purinergic receptors, also has a direct anticancer effect on human breast cancer cells, prostate carcinoma cells, human adenocarcinoma cells and fibroblast cell lines. Cytotoxic T lymphocytes and natural killer (NK) cells release ATP when they attack tumour cells [22]. Only transformed cell growth is inhibited, by inducing S phase block, apoptosis, increased permeability to nucleotides, sugar phosphates, ions and synergy with other anticancer agents. None of these effects are noted on untransformed cells [14].
Curiously, tumour cells are known to contain exceptionally high levels of ATP [23]. Adenosine and ATP both increase intratumour blood flow by stimulating nitric oxide synthesis from the endothelium, thus inducing potent vasodilution [24]. In this case ATP acts through P2Y receptors (26). Nitric oxide release is also linked to P2X receptor function. For instance, 90% of the nitric oxide synthase activity found in non-pregnant sheep myometrium is calcium ion-channel dependent [25].
Epithelial adhesive proteins also play a major role in the spread of cancer [26]. In wound healing, cell injury signals propagate via extracellular P2X receptors and intercellular gap junctions, stimulating calcium ion-induced wave propagation [27]. Intracellular calcium ions admitted by the P2X channels trigger the transport of membrane-bound organelles along microtubules, remodelling of the ECM and up-regulation of the adhesion molecule E-cadherin [28]. The myoepithelial cells found in prostatic epithelial acinar exert important paracrine effects on carcinoma cells both in situ and in vitro. Cancer cells are also affected by high expression of ECM molecules, proteinase inhibitors and angiogenic inhibitor [29]. During metastatic invasion, extracellular calcium influx activates membrane-associated metalloproteinases that facilitate tissue penetration by invasive cells. Urokinase plasminogen activator has also been strongly implicated in the progression of several malignancies including breast and prostate cancer [30].
Current techniques for staging and diagnosing cancer need to be improved in order to provide more reliable results using relatively simple technology. It would also be advantageous to have a diagnostic method amenable to automation.
It is an object of the present invention to provide a method of identifying pre-neoplastic and/or neoplastic cells which will overcome or substantially ameliorate at least some of the deficiencies of the prior art or will provide a useful alternative.